基于网络药理学和单细胞转录组探究灵芝蜂胶胶囊抗肝细胞癌的潜在机制

Potential mechanism of Ganoderma Lucidum Propolis Capsules against hepatocellular carcinoma based on network pharmacology and single cell transcriptome analysis

目的采用网络药理学和单细胞转录组分析方法研究灵芝蜂胶胶囊抗肝细胞癌的潜在机制。方法利用TCMSP、本草组鉴(HERB)和SwissTargetPrediction数据平台,获得灵芝和蜂胶的活性成分及靶点。基于TCGA数据库获取肝癌RNAseq数据,运用R软件包DESeq2进行差异分析,将差异基因与药物靶点的交集基因作为灵芝蜂胶胶囊抗肝细胞癌的潜在靶点基因。采用R软件包clusterProfilter进行GO和KEGG富集分析。利用Cox和KM方法对交集靶点进行筛选,再使用GEO数据库中的GSE14520进行生存分析再次筛选得到核心基因。利用分子对接进行核心靶点基因和对应成分的模拟结合。采用GSE149614数据对核心基因进行单细胞分析,并对关键基因在多种癌症中的生存预后进行分析。采用qPCR验证灵芝蜂胶对HepG2和Huh7细胞的干预作用。结果基于网络药理学成功鉴定出肝细胞癌差异基因706个,灵芝蜂胶潜在作用靶点603个,二者交集靶点29个。富集分析结果主要涉及神经活性配体-受体相互作用和细胞周期等。满足生存分析筛选的关键靶点为AURKB、CCNA2、CCNB1、CCR3、CDK1、PLK1、TOP2A和TTK。分子对接结果表明这些关键基因与对应成分均具有良好的结合能力。qPCR结果显示灵芝蜂胶可显著抑制HepG2和Huh7细胞中AURKB基因的表达。结论灵芝蜂胶可能通过过氧麦角甾醇等成分作用于AURKB等基因参与细胞周期等过程发挥抗肝细胞癌的作用,为进一步研究提供思路和参考。

Objective To investigate the potential mechanism of Ganoderma Lucidum Propolis(GLP)Capsules against hepatocellular carcinoma(HCC)by using network pharmacology and single cell transcriptome analysis.Methods The targets of GLP were obtained by TCMSP,HERB and SwissTargetPrediction data platforms.The liver cancer RNA-seq data were obtained from TCGA database,and the R software package DESeq2 was used for differential analysis.The intersection genes of the significant fold change genes and the drug target genes were used as potential target genes.GO and KEGG enrichment analyses were performed by the R package clusterProfilter.The Cox and KM methods were used to screen the intersection targets,and then GSE14520 in the GEO database was used for survival analysis to screen the core genes again.Molecular docking was used to simulate the combination of genes and their corresponding components.Single cell analysis of key genes was performed by using GSE149614 data.And the survival prognosis of the key genes was analyzed in multiple cancers.quantitative polymerase chain reaction(qPCR)was used to verify the intervention effect of GLP Capsules on HepG2 and Huh7 cells.Results Totally 706 HCC differential genes were successfully identified,603 were identified as potential targets of GLP,and 29 were identified as intersection targets.Enrichment analysis results mainly involved neuroactive ligand-receptor interaction and cell cycle.The key targets that met the survival analysis screening were AURKB,CCNA2,CCNB1,CCR3,CDK1,PLK1,TOP2A and TTK.Molecular docking showed that these key genes had good binding ability with their corresponding components.qPCR further verified that GLP significantly inhibited the expression of AURKB gene in HepG2 and Huh7 cells.Conclusion GLP may exert anti-HCC by acting on AURKB and other genes involved in cell cycle processes mainly through peroxyergosterol and other components,providing ideas and references for further research.